The Molecular Mechanism Of Drought Tolerance In PeaT1 Transgenic Rice

Immunity resistance is one of the crucial steps during the plant development.Elicitor,which could induce plant immunity response,has good application prospect in biological control.Thus,exploring the molecular mechanism of elicitor on stress tolerance not only offers reference for the analysis of the expression function of plant allogeneic gene but also has theoretical and practical significance on genetic improvement of plant breeding for stress tolerance.PeaT1 was isolated from Alernaria tenuissima by our lab previously and the homozygous PeaT1 transgenic rice lines(PeaT1OE)have been obtained.In the present study,the function of elicitor PeaT1 on drought resistance in rice was analyzed and the molecular mechanism was preliminarily elaborated.Results are as followed.(1)PeaT1 over-expressing plants increased drought tolerance under water deficiency in rice.After recovery,the survival rate was 20%higher than the wild type(WT).In PeaT1OE plants,abscisic acid,chlorophyll,and proline content were significantly increased,while the malondialdehyde(MDA)content prominently decreased compared with the wild-type plants.Drought related genes(OsCPK9,OsSKIPa,OsNCED2,OsNAC9,OSTPKb,and OsEREBP1)expression levels were higher in PeaT1OE than WT.(2)Proteins involved in stress resistance were highly expressed in PeaT1OE plants.Label free quantitative proteomic analysis between PeaT1OE and WT showed that a total of 4493 proteins were identified.Among them,57 proteins(32 for up-regulation and 25 for down-regulation)were significant changed.Combing GO analysis,these different proteins were mostly involved in metabolism,stress response,protein metabolism,cell activity,and biological processes.And the protein SKIP/SNW significantly increased its expression in PeaT1OE rice.(3)PeaT1 interacted with OsMIOX in vitro and in vivo.Yeast translation assay demonstrated that PeaT1 had self-activating effect,while it was inhibited under the 3-AT(50 mM/L).By yeast-two hybrid libray screening,OsMIOX protein,one of nine interaction protein could interact with PeaT1 in yeast,moreover,the interaction verified by BIFC and MBP pull down.(4)qRT-PCR and sub-location assays showed OsMIOX expressed highly in PeaT1OE rice during drought stress.The signal of in situ hybridization(ISH)was enriched in leaf mesophyll cells and anther.OsMIOX-GFP fluorescence was co-localized only with the endoplasmic reticulum(ER)marker HDEL.(5)By constructing the vector of overexpressing-OsMIOX and the crispr-OsMIOX,we finally obtained 10 positive overexpressing lines and 6 positive knocking out lines.Transcriptome analysis between the OsMIOX OE rice and the wild type during water deficiency at 7 days showed that 743 genes changed significantly,534 up-regulated and the others down-regulated.GO analysis showed that the main changed pathway was enriched in plant hormone signal ABA,JA and sugar metabolism.Taken together,PeaT1 improved rice drought tolerance by interacting with OsMIOX and several stess related genes were affected.This study not only offers theoretical reference but also has crucial significance on the genetic improvement of rice molecular breeding on drought tolerance.